Osteoporosis is a common disease mainly characterized by low bone mineral density (BMD) and increased risk of fractures. Peripheral blood monocytes (PBMs) may not only act as precursors of osteoclasts, the bone resorption cells, but also produce cytokines important for osteoclast differentiation, activation, and apoptosis, and thus represent major systemic cells for bone metabolism. Alterations in DNA methylation as an important epigenetic regulator of gene expression, is significant in the etiology of human complex diseases. In vitro studies have shown that DNA methylation is involved in osteoclastogenesis; however, the in vivo significance of global DNA methylation profiles (methylome) in humans underlying osteoporosis risk is unknown. Our Hypothesis is that altered DNA methylation profiles in PBMs and the associated changes in gene expression and osteoclastogenesis contribute to peak BMD variation in humans. Our Goal/Expectation is to i) identify differentially methylated regions (DMRs) in PBMs at the whole methylome level between premenopausal women with extremely high peak BMD and those with extremely low peak BMD; ii) study potential epigenetic mechanisms of osteoporosis, namely, how the DMRs identified may influence the peak BMD variation through affecting the expression of the relevant genes and subsequent osteoclastogenesis. Methods: 1) PBMs and their DNAs and total RNAs will be extracted from 160 premenopausal Caucasian females aged 25-40 years, including 80 with extremely high peak BMD and 80 with extremely low peak BMD (but otherwise matched). 2) DMRs will be identified by performing state-of-the-art methylome profiling studies with the cutting-edge technology MeDIP-seq (methylated DNA immunoprecipitation assays followed by next-generation sequencing) in a discovery sample of 80 subjects (including 40 with high and 40 with low BMD). 3) The identified DMRs will be subject to confirmation by bisulfite sequencing in an independent replication sample (including 40 with high and 40 with low BMD), and their target genes will be identified by correlating the DNA methylation data with the mRNA expression levels of the potential candidate target genes in PBMs of the total 160 subjects. 4) The roles of the identified most significant DMR-affiliated target genes on osteoclastogenesis will be further investigated by cell based in vitro assays. This highly novel R01 project holds great promise of award to generate breakthroughs in the osteoporosis research field. The results may lead to a major paradigm shift by expanding current genetic epidemiology studies of osteoporosis, from classical DNA variants to novel epigenetics/epigenomics mechanisms of DNA modification. Therefore, the results will be highly important for understanding the underlying molecular mechanisms, and thus help prevention and treatment, of osteoporosis.